ABSTRACT PURPOSE: Skin metastases of breast cancer remain a therapeutic challenge. Toll-like receptor 7 agonist imiquimod is an immune response modifier and can induce immune-mediated rejection of primary skin malignancies when topically applied. Here we tested the hypothesis that topical imiquimod stimulates local anti-tumor immunity and induces the regression of breast cancer skin metastases.EXPERIMENTAL DESIGN: A prospective clinical trial was designed to evaluate the local tumor response rate of breast cancer skin metastases treated with topical imiquimod, applied 5 days/week for 8 weeks. Safety and immunological correlates were secondary objectives.RESULTS: Ten patients were enrolled and completed the study. Imiquimod treatment was well tolerated, with only grade 1-2 transient local and systemic side effects consistent with imiquimod's immunomodulatory effects. Two patients achieved a partial response (20%; 95% CI 3% - 56%). Responders showed histological tumor regression with evidence of an immune-mediated response, demonstrated by changes in the tumor lymphocytic infiltrate and locally produced cytokines. CONCLUSIONS: Topical imiquimod is a beneficial treatment modality for breast cancer metastatic to skin/chest wall and is well tolerated. Importantly, imiquimod can promote a pro-immunogenic tumor microenvironment in breast cancer. Preclinical data generated by our group suggest even superior results with a combination of imiquimod and ionizing radiation and we are currently testing in patients whether the combination can further improve anti-tumor immune and clinical responses.

Updated Version 10.1158/1078-0432.CCR-12-1149doi:Access the most recent version of this article at: MaterialSupplementary mlhttp://clincancerres.aacrjournals.org/content/suppl/2012/07/05/1078-0432.CCR-12-1149.DC1.htAccess the most recent supplemental material at:ManuscriptAuthorbeen edited.Author manuscripts have been peer reviewed and accepted for publication but have not yet

Purpose: Skin metastases of breast cancer remain a therapeutic challenge. Toll-like receptor 7 agonist imiquimod is an immune response modifier and can induce immune-mediated rejection of primary skin malignancies when topically applied. Here we tested the hypothesis that topical imiquimod stimulates local anti-tumor immunity and induces the regression of breast cancer skin metastases. Methods: A prospective clinical trial was designed to evaluate the local tumor response rate of breast cancer skin metastases treated with topical imiquimod, applied 5 days/week for 8 weeks. Safety and immunological correlates were secondary objectives. Results: Ten patients were enrolled and completed the study. Imiquimod treatment was well tolerated, with only grade 1-2 transient local and systemic side effects consistent with imiquimod’s immunomodulatory effects. Two patients achieved a partial response (20%; 95% CI 3% - 56%). Responders showed histological tumor regression with evidence of an immune-mediated response, demonstrated by changes in the tumor lymphocytic infiltrate and locally produced cytokines. Conclusion: Topical imiquimod is a beneficial treatment modality for breast cancer metastatic to skin/chest wall and is well tolerated. Importantly, imiquimod can promote a pro-immunogenic tumor microenvironment in breast cancer. Preclinical data generated by our group suggest even superior results with a combination of imiquimod and ionizing radiation and we are currently testing in patients whether the combination can further improve anti-tumor immune and clinical responses.

INTRODUCTION Skin metastases of solid tumors remain a therapeutic challenge. Breast cancer is the second most common tumor, after melanoma, to metastasize to the skin [1, 2]. Breast cancer skin recurrence most frequently manifest after mastectomy and can present as firm nodules, diffuse infiltration or ulcerative lesions, often in proximity of the mastectomy scar. Initial management of recurrences usually includes resection and radiation, but skin metastases tend to recur and herald diffuse metastatic spread. Furthermore, cutaneous metastases affect quality of life and become a debilitating experience for the patient as progression of disease leads to chest wall ulceration, bleeding and super-infection. Therefore, novel treatment approaches are warranted.

Based on imiquimod’s efficacy in primary skin tumors and encouraged by anecdoctal reports of anti-tumor efficacy in skin metastases of melanoma and breast cancer [9, 10], we tested the hypothesis that treatment with topical imiquimod could induce the regression of breast cancer skin metastases. In a prospective phase II trial topical imiquimod 5% was applied to all cutaneous metastases and local anti-tumor activity and toxicity were measured after an 8-week treatment course. Tumor punch-biopsies were obtained before and after imiquimod treatment from each patient to study the immunological changes in the tumor microenvironment.

completion of prior radiotherapy and hyperthermia to the target area >4 weeks and >10 weeks respectively, prior to study entry. Systemic disease assessment by CT/PET-CT imaging pre- and post-treatment was not required by protocol and was left to the discretion of the treating physician. All patients provided a written informed consent for participation in this IRB-approved study (clinicaltrials.gov identifier NCT00899574).

Trial design The primary objective of this trial was to determine the local anti-tumor effect of topical TLR7 agonist imiquimod 5% cream in breast cancer patients with skin metastases. Secondary objectives were to assess toxicity and to study the immunological effects in the tumor microenvironment induced by imiquimod treatment. The trial was designed as an open label, single arm study to test the null hypothesis that the local anti-tumor effect (CCR and PR) was P<0.05 versus the alternative that P>0.20. An optimal two-stage Simon design was used, in which 10 patients were to be enrolled in stage one, with an expansion to stage 2 with an additional 19 patients if there was at least one responder in stage 1. The overall alpha level for this design was 0.047 with power of 0.801. At study entry, patient demographic and tumor characteristics (pathology, grade of differentiation, hormone receptor and human epidermal growth factor receptor (Her)-2 status), metastatic sites and treatment history were collected.

metastases for 5 days/week for 8 weeks (one cycle). Additional treatment cycles were left to the discretion of patient and treating physician. The cream was thinly spread onto the lesions, remained on the skin for approximately 8 hours overnight, and was washed off the following morning. One single use packet (containing 250 mg of the cream) was used to cover areas up to 100 cm2; another packet was used for each additional treatment area of 100cm2, up to a maximum of 6 packets per day. These dose determinations were based on extrapolation from clinical experience with dosing of up to 6 packets per application in patients with actinic keratoses [11, 12]. Imiquimod application was recorded by means of patient diaries and compliance was encouraged and monitored by weekly phone calls of study personnel to patients.

(PR, residual disease less than 50% of original tumor size), stable disease (SD, 50-99% of original tumor size), no response (NR, 100-124% of original tumor size) and progressive disease (PD, 125% or greater of original tumor size or new skin lesions).

flow cytometry. Cells were surface stained with the following antibodies: CD3-PerCP-Cy5.5, CD4-Alexa700 or PE, CD8-Pacific blue, CD25-PE, CD45RO-APC (Biolegend, San Diego, CA), CCR7-FITC (R&D Systems, Minneapolis, MN) and CCR6-biotin with Streptavidin-APC (BD Pharmingen, San Diego, CA). For FoxP3 staining, surface staining was followed by intracellular staining using the FoxP3 Staining buffer set (Ebiosciences) and FoxP3-Alexa488 antibody (Biolegend). For intracellular cytokine staining, cells were stimulated for 5 hours at 37oC with PMA 20ng/mL and Ionomycin 500ng/mL (Sigma-Aldrich, St Louis, MO) and Golgistop (BD Biosciences). Cells were then fixed and permeabilized using the same FoxP3 Staining buffer set (eBioscience) and stained with IFNγ-PeCy7 and IL-4-APC (Ebiosciences). Stained samples were acquired on an LSRII flow cytometer (BD Pharmingen). Flow cytometry data were analyzed using FlowJo software (version 8.8.7, Tree-Star Inc., Ashland, OR).

Statistical analysis Characteristics of patients are summarized using descriptive statistics including median and ranges for continuous variables and frequencies for categorical variables. Response rates (CCR+PR) were estimated at the conclusion of the first stage of the trial along with exact 95% confidence intervals. Safety data was summarized by body system and type and most severe Common Terminology Criteria for Adverse Events (CTCAE version 3.0) grade of individual events at the patient level. Changes in tumor supernatant cytokine values from pre-treatment to post-treatment were evaluated using Wilcoxon non-parametric signed rank tests (2-sided).

RESULTS Ten women enrolled and completed the first stage of this two stage study. The median age was 50 years. Demographic and tumor characteristics as well as treatment history are shown in Table 1. Seven women presented with a chest wall recurrence, and 3 women presented with skin involvement of a large primary breast cancer in the setting of systemic metastases. All women had failed prior treatment for metastatic/recurrent disease, ranging from 1-3 lines of hormonal therapy (average 2) and 1-5 lines of chemotherapy (average 2.5). Based on the skin area involved, six patients applied 1 packet per day, whereas 4 applied more than 1 packet per day. A second treatment cycle was administered in 2 patients. Patient compliance, defined as the number of administered applications divided by the number of prescribed applications during the entire study period, was excellent with 4 patients not missing any doses, and 6 patients having a compliance score of 95% or greater (1-2 missed doses).

treatment, similar to symptoms observed with systemic interferon alpha treatment [14]. The increase of intratumoral as well as circulating IFN-α2 concentrations (from 7 to 19 pg/ml in plasma) with imiquimod treatment in this patient suggests a systemic spillover effect of locally induced cytokines. The most frequently observed AEs were local, at the application site, and were experienced by 7 of 10 patients (Table 2). Symptoms included itching, burning and pain at the target site while signs included erythema, desquamation and infection. Topical antibiotics were administered for superficial infection at the treatment site, as indicated. Patient discomfort due to local or systemic AEs, regardless of grade, was successfully managed with temporary dosing interruptions (one patient for 3 weeks) and subsequent reduction of the application frequency from 5x to 3x per week (three patients).

achieved response rate of 20% with its 95%CI was reassuring that imiquimod as single agent has efficacy.

Interestingly, two of the 10 patients treated in the present study (both with local SD on study) experienced a complete clinical remission upon treatment with a subsequent systemic regimen (fulvestrant). In both women the complete remission in the skin lesion was associated with a systemic complete response (pulmonary and osseous in one patient, mediastinal lymph node and adrenal metastases in the second patient) and have been maintained for over one year (details are being reported separately).

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[Show abstract][Hide abstract]ABSTRACT: Toll-like receptors (TLRs) have first been characterized for their capacity to detect conserved microbial components like lipopolysaccharide (LPS) and double-stranded RNA, resulting in the elicitation of potent (innate) immune responses against invading pathogens. More recently, TLRs have also been shown to promote the activation of the cognate immune system against cancer cells. Today, only three TLR agonists are approved by FDA for use in humans: the bacillus Calmette-Guérin (BCG), monophosphoryl lipid A (MPL) and imiquimod. BCG (an attenuated strain of Mycobacterium bovis) is mainly used as a vaccine against tuberculosis, but also for the immunotherapy of in situ bladder carcinoma. MPL (derived from the LPS of Salmonella minnesota) is included in the formulation of Cervarix®, a vaccine against human papillomavirus-16 and -18. Imiquimod (a synthetic imidazoquinoline) is routinely employed for actinic keratosis, superficial basal cell carcinoma, and external genital warts (condylomata acuminata). In this Trial Watch, we will summarize the results of recently completed clinical trials and discuss the progress of ongoing studies that have evaluated/are evaluating FDA-approved TLR agonists as off-label medications for cancer therapy.

[Show abstract][Hide abstract]ABSTRACT: PURPOSE: This study tested the hypothesis that topical Toll-Like Receptor (TLR) 7 agonist imiquimod promotes anti-tumor immunity and synergizes with other treatments in a model of skin-involving breast cancer. EXPERIMENTAL DESIGN: TSA mouse breast carcinoma cells were injected s.c. into syngeneic mice. Imiquimod 5% or placebo cream was applied topically on the shaved skin overlying tumors three times/week. In some experiments, local ionizing radiation therapy (RT) was delivered to the tumor in 3 fractions of 8 Gy, given on consecutive days. Cyclophosphamide (CY) was given i.p. in one dose of 2 mg/mouse. Mice were followed for tumor growth and survival. RESULTS: Treatment with imiquimod significantly inhibited tumor growth, an effect that was associated with increased tumor infiltration by CD11c+, CD4+ and CD8+ cells, and abolished by depletion of CD8+ cells. Administration of imiquimod in combination with RT enhanced significantly tumor response compared to either treatment alone (p<0.005), and 11 to 66% of irradiated tumors completely regressed. Importantly, the addition of topical imiquimod also resulted in growth inhibition of a secondary tumor outside of the radiation field. Low dose CY given before start of treatment with imiquimod and RT further improved tumor inhibition and reduced tumor recurrence. Mice that remained tumor-free rejected a tumorigenic inoculum of TSA cells, demonstrating long-term immunological memory. CONCLUSIONS: Topical imiquimod inhibits tumor growth and synergizes with RT. Addition of CY further increases the therapeutic effect and induces protective immunological memory, suggesting that this combination is a promising strategy for cutaneous breast cancer metastases.

[Show abstract][Hide abstract]ABSTRACT: Over the prior two decades, imiquimod, a toll-like receptor 7 agonist, has been applied to nearly fifty clinical settings. Due to its immunomodulatory role, the topical cream today for the first time, is being applied to cutaneous breast cancer in pre-clinical models and in a Phase 2 clinical trial.